This invention relates to improvements in processes for conducting “sandwich” assays of proteins and, in particular to an improved method for simultaneously detecting and/or determining, a plurality of modified proteins, especially modified cellular proteins, in a sample. In a typical “sandwich” assay for proteins, a sample (generally a biological fluid or cell extract) comprising a mixture of materials, including one or more proteins, is contacted with a series of microparticles or beads associated with antibodies that bind to the respective target proteins. Each combination of microparticles with a particular antibody has a feature, for instance color, that can distinguish it from the others. After washing away unbound materials, the microparticles, now containing proteins bound to the antibodies, are contacted with a second antibody that binds specifically to a particular protein to be detected. The second antibody typically includes a means for its detection, such as a fluorescent marker. The target protein is detected, and the quantity determined, by means suitable to the situation (e.g. scanning for fluorescence).
Most cellular proteins require modifications in order to transfer from their silent state to the active form. Such modifications include phosphorylation (for instance, on tyrosine, threonine and/or serine residues), glycosylation (on membrane proteins), and acetylation, prenylation and methylation (on lysine residues). Detection and measurement of such modified proteins, as currently carried out, is limited to processes that involve multiple steps but that can determine only a single modified protein in each assay. For instance, to investigate phosphorylation of proteins, current procedures use 32P-labeled inorganic phosphate in combination with immunoprecipitation and SDS-PAGE autoradiography or Western blot analyses specific for the phosphorylated form of the target protein. However Western blot processes for this purpose are slow and time-consuming and can only determine one analyte at a time.
A method for simultaneously detecting and, if desired, determining, two or more modified proteins in a sample, in a single operation, would be desirable.